Tissue factor pathway inhibitor-3

ABSTRACT

The present invention relates to a novel TFPI-3 protein which is a member of the tissue factor protease inhibitor family. In particular, isolated nucleic acid molecules are provided encoding human TFPI-3 proteins. TFPI-3 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TFPI-3 activity. Also provided are diagnostic methods for detecting hemostasis system-related disorders and therapeutic methods for treating hemostatis system-related disorders.

This application claims benefit of 35 U.S.C. section 119(e) based onU.S. Provisional Application Serial No. 60/036,703, filed Jan. 31, 1997,and is incorporated herein by reference.

The present invention relates to a novel human gene encoding apolypeptide which is a member of the Kunitz-type protease inhibitorfamily. More specifically, isolated nucleic acid molecules are providedencoding a human polypeptide named Tissue Factor Pathway Inhibitor-3hereinafter referred to TFPI-3. TFPI-3 polypeptides are also provided,as are vectors, host cells and recombinant methods for producing thesame. Also provided are diagnostic methods for detecting disordersrelated to vascular hemostatsis and therapeutic methods for treatingsuch disorders. The invention further relates to screening methods foridentifying agonists and antagonists of TFPI-3.

BACKGROUND OF THE INVENTION

Proteases are responsible, either directly or indirectly, for all bodilyfunctions, including cell growth, differentiation and death (apoptosis),cell nutrition, intra- and extracellular protein turnover (housekeepingand repair), cell migration and invasion, and fertilization andimplantation. These functions extend from the cellular level to theorgan and organism level to produce cascade systems such as hemostatisand inflamation, and complex processes at all levels of physiology andpathophysiology.

Maintenance of vascular integrity is an important host response toinjury. Complex hemostatis mechanisms of coagulation, platelet function,and fibrinolysis exist to minimize adverse consequences of vascularinjury and to accelerate vascular repair. Many of these hemostaticmechanisms are initiated and/or regulated by cells of the wall of theblood vessel.

Tissue-factor-pathway inhibitor (TFPI) is a cell-surface associatedglycoprotein which plays a key role in the regulation of tissuefactor-initiated blood coagulation. Human TFPI is a trace 42-kDa plasmaglycoprotein that is synthesized primarily by endothelial cells andconsists of a negatively charged amino terminal region, three tandemKunitz-type inhibitor domains, and a highly basic carboxyl-terminal tail(Wun, T. C., et al., J. Biol. Chem. 263:6001 (1988)). After a 22-residuesignal peptide, the mature protein contains 213 amino acids with 18cysteines. TFPI forms a complex with factor Xa and inhibits itsamidolytic and proteolytic activity. The factor Xa-TFPI complex rapidlyinhibits activity of the factor VIIa-tissue factor complex.

The cloning and characterization of a gene coding for a second tissuefactor pathway inhibitor (TFPI-2), has been reported (Sprecher, C. A. etal., Proc. Natl. Acad, Sci. USA, 91, 3353-3357 (1994)). This gene wasinitially identified on the basis of primary sequence homology andstructural similarity. Subsequent characterization has confirmed itspredicted activity as a protease inhibitor. Alterations of thehemostatic system can result from such causes as neoplasia and trauma.Such alterations result in an increased incidence of thromboticdisorders such as venous thrombosis, pulmonary embolism, atrialfibrillation, cerebral thrombosis, and hemophilia. Thus, there is a needfor identification and characterization of polypeptides that function asinhibitors of the coagulation pathway which can play a role indetecting, preventing, ameliorating or correcting such disorders.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the TFPI-3polypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by the cDNA clone depositedas plasmid DNA as ATCC Deposit Number 97797 on Nov. 20, 1996. Thenucleotide sequence determined by sequencing the deposited TFPI-3 clone,which is shown in SEQ ID NO:1, contains an open reading frame encoding acomplete polypeptide of 252 amino acid residues, including an initiationcodon encoding an N-terminal methionine at nucleotide positions 361 to363 and a predicted molecular weight of about 28.2 kDa.

The TFPI-3 protein of the present invention shares sequence homologywith the translation product of the human MRNA for Tissue Factor PathwayInhibitor (TFPI), TFPI-2 and aprotinin, including the followingconserved domains: (a) a first predicted Kunitz-type domain of about 51amino acids; and (b) a second predicted Kunitz-type domain also of about51 amino acids, both of which are thought to be important in regulatingblood coagulation, and are underscored with stars in FIGS. 1A and 1B.The homology between human TFPI, TFPI-2, aprotinin and TFPI-3, shown inFIG. 2, indicates that TFPI-3 is also a protease and may be involved inregulating blood coagulation. Protease inhibition assays describedherein confirm the ability of TFPI-3 polypeptides to inhibit proteaseactivity.

The encoded polypeptide has a predicted leader sequence of 27 aminoacids underlined in FIGS. 1A and 1B; and the amino acid sequence of thepredicted mature TFPI-3 protein is also shown in FIG. 1, and as aminoacid residues 1-225 (SEQ ID NO:2).

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a polypeptide comprising the predicted second Kunitz-typedomain of the TFPI-3 polypeptide having the amino acid sequence atpositions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clonecontained in ATCC Deposit No. 97797; (b) a nucleotide sequence encodinga polypeptide comprising the consensus Kunitz-type domain having theamino acid sequence shown as SEQ ID NO:28; and (c) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a) or (b) above;wherein said nucleic acid sequence in (a) or (b) does not encode apolypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30,or SEQ ID NO:31.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b) or (c),above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b) or (c), above.This polynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a TFPI-3polypeptide having an amino acid sequence in (a) or (b), above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTFPI-3 polypeptides or peptides by recombinant techniques.

The invention further provides an isolated TFPI-3 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the polypeptide comprising the predicted secondKunitz-type domain of the TFPI-3 polypeptide having the amino acidsequence at positions 106 to 156 in SEQ ID NO:2 or as encoded by thecDNA clone contained in ATCC Deposit No. 97797; or (b) the amino acidsequence of the polypeptide comprising the consensus Kunitz-type domainhaving the amino acid sequence of SEQ ID NO:28; wherein said nucleicacid sequence in (a) or (b) does not encode a polypeptide comprising asequence shown as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence at least 80% identical, more preferably atleast 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a) or (b) above, as well aspolypeptides having an amino acid sequence with at least 90% similarity,and more preferably at least 95% similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises the amino acid sequence of anepitope-bearing portion of a TFPI-3 polypeptide having an amino acidsequence described in (a) or (b), above. Peptides or polypeptides havingthe amino acid sequence of an epitope-bearing portion of a TFPI-3polypeptide of the invention include portions of such polypeptides withat least six or seven, preferably at least nine, and more preferably atleast about 30 amino acids to about 50 amino acids, althoughepitope-bearing polypeptides of any length up to and including theentire amino acid sequence of a polypeptide of the invention describedabove also are included in the invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a TFPI-3 polypeptide having an amino acid sequencedescribed in (a) or (b) above. The invention further provides methodsfor isolating antibodies that bind specifically to a TFPI-3 polypeptidehaving an amino acid sequence as described herein. Such antibodies areuseful diagnostically or therapeutically as described below.

The invention also provides for pharmaceutical compositions comprisingTFPI-3 polypeptides, particularly human TFPI-3 polypeptides, which maybe employed, for instance, in inhibiting intravascular clotting andpreventing the formation of fribrin clots both in vitro and in vivo, foranticoagulant therapy in prophylaxis of venous thrombosis and astreatment for preventing its extension, as well as to provide low-doseregiment for prevention of postoperative deep venous thrombosis andpulmonary embolism, for the prophlaxis and treatment of pulmonaryembolism and atrial fibrillation with embolism, to prevent clotting inarterial and heart surgery as well as for prevention of cerebralthrombosis in evolving stroke, for treating coronary occlusion withacute myocardial infarction and in the prophylaxis and treatment ofperipheral arterial emoblism, for the treatment of sepsis, inflamatorydiseases and transplant rejection, in the treatment of hyperfibrinolytichemorrhage and traumatic hemorrhagic shock as well as in diseasesconnected with excessive release of pancreatic elastase (pancreatitis),serum elastase (artherosclerosis), leukocyte elastase in acute andchronic inflammation with damage to connective tissue, in damage tovessel walls, in necrotic diseases, and in degeneration of lung tissue.Methods of treating individuals in need of TFPI-3 polypeptides are alsoprovided.

The invention further provides compositions comprising a TFPI-3polynucleotide or a TFPI-3 polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise a TFPI-3 polynucleotide forexpression of a TFPI-3 polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of a TFPI-3.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe TFPI-3 polypeptide, which involves contacting a protease which isinhibited by TFPI-3 polypeptide with the candidate compound in thepresence of a TFPI-3 polypeptide and a substrate cleavable by theselected protease, assaying the inhibitory activity of the proteaseactivity of the protease in the presence of the candidate compound andof TFPI-3 polypeptide, and comparing the protease activity to a standardlevel of activity, the standard being assayed when contact is madebetween the protease and substrate in the presence of the TFPI-3polypeptide, and in the absence of the candidate compound. In thisassay, an increase in inhibitory activity over the standard indicatesthat the candidate compound is an agonist of TFPI-3 activity and adecrease in inhibitory activity compared to the standard indicates thatthe compound is an antagonist of TFPI-3 activity.

It has been discovered that TFPI-3 is expressed at differing levels insome tissues. Therefore, nucleic acids of the invention are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). In addition, for a number of disorders of the abovetissues or cells, particularly of the hemostatic system, significantlyhigher or lower levels of TFPI-3 gene expression may be detected incertain tissues (e.g., cancerous and wounded tissues) or bodily fluids(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” TFPI-3gene expression level, i.e., the TFPI-3 expression level in healthytissue from an individual not having the hemostatic system disorder.Thus, the invention provides a diagnostic method useful during diagnosisof such a disorder, which involves: (a) assaying TFPI-3 gene expressionlevel in cells or body fluid of an individual; (b) comparing the TFPI-3gene expression level with a standard TFPI-3 gene expression level,whereby an increase or decrease in the assayed TFPI-3 gene expressionlevel compared to the standard expression level is indicative ofdisorder in the hemostatic.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of TFPI-3 activityin the body comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an isolated TFPI-3polypeptide of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of TFPI-3 activityin the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of an TFPI-3antagonist. Preferred antagonists for use in the present invention areTFPI-3-specific antibodies and TFPI-3 proteins having an amino acidresidue other than arginine at the P1 residue of either the first orsecond Kunitz-type domain, positions 21 and 116 of SEQ ID NO:2,respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the nucleotide sequence (SEQ ID NO:1) and deducedamino acid sequence (SEQ ID NO:2) of TFPI-3. The predicted leadersequence of about 27 amino acids is underlined. The leader sequence haspositions −27 to −1 in SEQ ID NO:2. The first Kunitz-type domain(Kunitz-1) has positions 11-61 in SEQ ID NO:2 and the second Kunitz-typedomain (Kunitz-2) has positions 106 to 156 in SEQ ID NO:2, both of whichare underscored with stars in FIGS. 1A and 1B.

FIG. 2 shows the regions of identity between the amino acid sequences ofthe Kunitz-type domains of TFPI-3 (Kunitz-1 SEQ ID NO:10, Kunitz-2 SEQID NO:11), the Kunitz-type domains of TFPI (Kunitz-1 SEQ ID NO:4,Kunitz-2 SEQ ID NO:5, Kunitz-3 SEQ ID NO:6), the Kunitz-type domains ofTFPI-2 (Kunitz-1 SEQ ID NO:7, Kunitz-2 SEQ ID NO:8, Kunitz-3 SEQ IDNO:9) and aprotinin (SEQ ID NO:11), as determined by the Clustal methodusing the computer program “Megalign” contained in the DNAStar suite ofprograms. This program generates a consensus sequence; i.e., a consensusKunitz-type domain, which is shown as SEQ ID NO:28.

FIG. 3 shows an analysis of the TFPI-3 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index—Jameson-Wolf” graph, the positive peaksindicate locations of the highly antigenic regions of the TFPI-3protein, i.e., regions from which epitope-bearing peptides of theinvention can be obtained.

FIG. 4 shows the results of the trypsin inhibition assay described inExample 5. The error bar represent standard deviation (n=4). TFPI-3-1refers to the first Kunitz-type domain of TFPI-3.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a Tissue Factor Pathway Inhibitor-3(“TFPI-3”) polypeptide having the amino acid sequence shown in SEQ IDNO:2, which was determined by sequencing a cloned cDNA. The nucleotidesequence shown in SEQ ID NO:1 was obtained by sequencing the HOEBN05clone, which was deposited on Nov. 20, 1996 at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 20852,and given accession number ATCC 97797. The deposited clone is containedin the pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.).

The TFPI-3 protein of the present invention shares sequence homologywith the translation product of the human mRNAs for TFPI, TFPI-2 andaprotinin. More specifically, the TFPI-3 contains two Kunitz typeprotease inhibitor domains which share striking similarity to theKunitz-type domains from proteases TFPI, TFPI-2 and aprotinin as can beseen in FIG. 2, including nearly perfect conservation among all sixcysteines contained in each. TFPI and TFPI-2 are thought to be importantprotease inhibitors acting to mediate hemostatis through an interactionwith Factor Xa and inhibition of the Factor VIIa-tissue factor complexin the blood coagulation cascade. Aprotinin likewise is an importantprotease inhibitor which has become a valuable drug, named Tyrasylol-®,for treatment of various diseases like, e.g., hyperfibrinolytichemmorrhage and traumatic-hemorrhagic shock (Fritz, H. et al., DrugRes., 33:479-494 (1983), and see, for example, U.S. Pat. No. 4,894,439).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A and 1B (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding a TFPI-3 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIGS. 1A and 1B (SEQ ID NO:1) was discoveredin a cDNA library derived from osteoblasts. Additional clones of thesame gene were also identified in cDNA libraries from the followingtissues: human fetal brain, fetal kidney, placenta, pituitary, testis,testis tumor, pancreas tumor, and macrophage.

The determined nucleotide sequence of the TFPI-3 cDNA of FIGS. 1A and 1B(SEQ ID NO:1) contains an open reading frame encoding a protein of 252amino acid residues, with an initiation codon at nucleotide positions361 to 363 of the nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO:1),and a deduced molecular weight of about 28.2 kDa. The amino acidsequence of the TFPI-3 protein shown in SEQ ID NO:2 is about 24%identical to human mRNA for TFPI-2 (Sprecher, C. A. et al., PNAS USA,91:3353 (1994), which can be accessed on GenBank as Accession No.L27624).

As mentioned above, the open reading frame of the TFPI-3 gene sharessequence homology with the translation product of the human mRNAs forTFPI, TFPI-2 and aprotinin (FIG. 2), including the following conserveddomains: (a) a first Kunitz-type domain of about 51 amino acids; and (b)a second Kunitz-type domain also of about 51 amino acids. The homologybetween TFPI, TFPI-2, aprotinin and TFPI-3 indicates that TFPI-3 mayalso be a protease inhibitor. Experiments described in Example 5 with arecombinantly cloned first Kunitz-type domain of TFPI-3 (constructionshown in Example 1), have shown that TFPI-3 polypeptides have theability to inhibit protease activity. Taken together this data indicatesthat TFPI-3 has protease inhibiting activity and may be important inregulation of blood coagulation.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete TFPI-3polypeptide encoded by the deposited cDNA, which comprises about 252amino acids, may be somewhat longer or shorter. More generally, theactual open reading frame may be anywhere in the range of ±20 aminoacids, more likely in the range of ±10 amino acids, of that predictedfrom the first methionine codon from the N-terminus shown in FIGS. 1Aand 1B (SEQ ID NO:1). It will further be appreciated that, depending onthe analytical criteria used for identifying various functional domains,the exact “address” of the Kunitz-type domains of the TFPI-3 polypeptidemay differ slightly from the predicted positions above. For example, theexact location of the TFPI-3 Kunitz-type domains in SEQ ID NO:2 may varyslightly (e.g., the address may “shift” by about 1 to about 10 residues,more likely about 1 to about 5 residues) depending on the criteria usedto define the domain. In this case, the ends of the Kunitz-type domainswere predicted on the basis of similarity between the TFPI-3 amino acidsequence and the sequence of several other mammalian proteases, and inparticular on the basis of the six conserved cysteines contained withineach domain.

Leader and Mature Sequences

The amino acid sequence of the complete TFPI-3 protein includes a leadersequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the TFPI-3 protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding the matureTFPI-3 polypeptide having the amino acid sequence encoded by the cDNAclone contained in the host identified as ATCC Deposit No. 97797. By the“mature TFPI-3 polypeptide having the amino acid sequence encoded by thecDNA clone in ATCC Deposit No. 97797” is meant the mature form(s) of theTFPI-3 protein produced by expression in a mammalian cell (e.g., COScells, as described below) of the complete open reading frame encoded bythe human DNA sequence of the clone contained in the vector in thedeposited host.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completeTFPI-3 polypeptide was analyzed by a computer program “PSORT”, availablefrom Dr. Kenta Nakai of the Institute for Chemical Research, KyotoUniversity (see K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)),which is an expert system for predicting the cellular location of aprotein based on the amino acid sequence. As part of this computationalprediction of localization, the methods of McGeoch and von Heinje areincorporated. The analysis of the TFPI-3 amino acid sequence by thisprogram predicted one cleavage site within the complete amino acidsequence shown in SEQ ID NO:2.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 361 to 363 of the nucleotide sequence shown in FIGS.1A and 1B (SEQ ID NO:1).

Also included are DNA molecules comprising the coding sequence for thepredicted mature TFPI-3 protein shown at positions 442-1116 of SEQ IDNO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode the TFPI-3 protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the TFPI-3 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97797 on Nov. 20, 1996. Preferably, this nucleic acidmolecule will encode the mature polypeptide, or a soluble extracellularform of the polypeptide containing one or both Kunitz-type domains (butlacking the transmembrane portion, about amino acids 196 to 225 in SEQID NO:2), encoded by the above-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) or thenucleotide sequence of the TFPI-3 cDNA contained in the above-describeddeposited clone, or a nucleic acid molecule having a sequencecomplementary to one of the above sequences. Such isolated molecules,particularly DNA molecules, are useful as probes for gene mapping, by insitu hybridization with chromosomes, and for detecting expression of theTFPI-3 gene in human tissue, for instance, by Northern blot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 442-1116 of SEQ ID NO:1.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HFCBP02R(SEQ ID NO:12), HKDH23F (SEQ ID NO:13), HPLBH53R (SEQ ID NO:14),HPLBH50R (SEQ ID NO:15), HCOSD62R (SEQ ID NO:16), HEPAB48R (SEQ IDNO:17), HAUAR79R (SEQ ID NO:18), and HPTTL69R (SEQ ID NO:19).

Polypeptides related to SEQ ID NO:2 include: Ala Asp Arg Glu Arg Ser IleHis Asp Phe Xaa Leu Val Ser Lys (SEQ ID NO:29); Lys Val Val Gly Arg XaaArg Ala Ser Met Pro Arg Trp Trp Tyr Asn Val Thr Asp Gly Ser Xaa Gln LeuPhe Val Tyr Gly Gly (SEQ ID NO:30); and Ala Thr Val Thr Glu Asn Ala ThrGly Asp Leu Ala Thr Ser Arg Asn Ala Ala Asp Ser Ser Val Pro Ser Ala Pro(SEQ ID NO:31).

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from nucleotide 361 to 1116. The inventionpreferably includes a polynucleotide comprising any portion of at leastabout 30 nucleotides, preferably at least about 50 nucleotides, of SEQID NO:1 from nucleotide 442 to 910. The invention most preferablyincludes a polynucleotide comprising any portion of at least about 30nucleotides, preferably at least about 50 nucleotides, of SEQ ID NO:1from nucleotide 442 to 631.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIGS. 1A and 1B (SEQ ID NO:1) is intended fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-300 nt in length arealso useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in FIGS. 1A and 1B (SEQ ID NO:1). By afragment at least 20 nt in length, for example, is intended fragmentswhich include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in FIGS. 1Aand 1B (SEQ ID NO:1). Preferred nucleic acid fragments of the presentinvention include nucleic acid molecules encoding epitope-bearingportions of the TFPI-3 polypeptide as identified in FIG. 3 and describedin more detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit No. 97797. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 (e.g., 50) nt of the reference polynucleotide. These areuseful as diagnostic probes and primers as discussed above and in moredetail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A and 1B (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the TFPI-3 cDNA shownin FIGS. 1A and 1B (SEQ ID NO:1)), or to a complementary stretch of T(or U) residues, would not be included in a polynucleotide of theinvention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode a TFPI-3 polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 27 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro- protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the TFPI-3 fusedto Fc at the N- or C-terminus.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the TFPI-3 protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the TFPI-3 protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 or themature TFPI-3 amino acid sequence encoded by the deposited cDNA clone.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a polypeptide comprising the predicted second Kunitz-typedomain of the TFPI-3 polypeptide having the amino acid sequence atpositions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clonecontained in ATCC Deposit No. 97797; (b) a nucleotide sequence encodinga polypeptide comprising the consensus Kunitz-type domain having theamino acid sequence shown in SEQ ID NO:28; and (c) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a) or (b), above;except that said polynucleotide of (a) or (b) does not encode apolypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30,or SEQ ID NO:31

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b) or (c),above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b) or (c), above.This polynucleotide which hybridizes does not hybridize under stringenthybridization conditions to a polynucleotide having a nucleotidesequence consisting of only A residues or of only T residues. Anadditional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a TFPI-3polypeptide having an amino acid sequence in (a) or (b), above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTFPI-3 polypeptides or peptides by recombinant techniques.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TFPI-3polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the TFPI-3polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIGS. 1A and 1B or to the nucleotidessequence of the deposited cDNA clone can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A and 1B (SEQ ID NO:1) or to the nucleic acid sequenceof the deposited cDNA, irrespective of whether they encode a polypeptidehaving TFPI-3 activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having TFPI-3 activity, oneof skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having TFPI-3 activityinclude, inter alia, (1) isolating the TFPI-3 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe TFPI-3 gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting TFPI-3 MRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIGS. 1A and 1B (SEQ ID NO:1) or to the nucleic acid sequenceof the deposited cDNA which do, in fact, encode a polypeptide havingTFPI-3 protein activity. By “a polypeptide having TFPI-3 activity” isintended polypeptides exhibiting activity similar, but not necessarilyidentical, to an activity of the mature TFPI-3 protein of the invention,as measured in a particular biological assay. For example, the TFPI-3protein of the present invention inhibits the protease activity oftrypsin and the amidolytic activity of factor VIIa-tissue factor. An invitro assay for measuring the trypsin inhibitory activity of TFPI-3 isdescribed in the literature, for example, in Sprecher, C. A. et al.,Proc. Natl. Acad, Sci. USA, 91, 3353-3357 (1994). Briefly, the assayinvolves coincubating TFPI-3 with trypsin and subsequently measuringresidual protease activity by measuring the activity of a chromogenicsubstrate. Such activity is useful for preventing the coagulation ofblood. Other such assays are known to those of skill in the art, forexample, as disclosed on page 13 in U.S. Pat. No. 4,894,436,incorporated herein by reference.

TFPI-3 protein modulates protease activity in a dose-dependent manner inthe above-described assay. Thus, “a polypeptide having TFPI-3 proteinactivity” includes polypeptides that also exhibit any of the sameprotease inhibitory activities in the above-described assays in adose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the TFPI-3 protein, preferably, “apolypeptide having TFPI-3 protein activity” will exhibit substantiallysimilar dose-dependence in a given activity as compared to the TFPI-3protein (i.e., the candidate polypeptide will exhibit greater activityor not more than about 25-fold less and, preferably, not more than abouttenfold less activity relative to the reference TFPI-3 protein). TFPI-3polypeptides may also be assayed for activity according to the assaydescribed in Example 5, below.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:1) willencode a polypeptide “having TFPI-3 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving TFPI-3 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of TFPI-3polypeptides or fragments thereof by recombinant techniques. The vectormay be, for example, a phage, plasmid, viral or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and K.Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

The TFPI-3 protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Polypeptides and Fragments

The invention further provides an isolated TFPI-3 polypeptide having theamino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2, or a peptide or polypeptide comprising aportion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of TFPI-3 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Holst et al., Thrombosis and Haemostasis,71(2):214-19 (1994) reported a modified TFPI protein that retainedantithrombotic activity despite containing only the first 161 aminoacids of a 276 amino acid protein. In the present case, since theprotein of the invention is a member of the TFPI polypeptide family,deletions of N-terminal amino acids up to the cysteine at position 106(C 106) of SEQ ID NO:2 may retain some biological activity such asprotease inhibitor activity. Polypeptides having further N-terminaldeletions including the C106 residue in SEQ ID NO:2 would not beexpected to retain biological activity of the second Kunitz-type domainbecause it is known that this residue in a TFPI-related polypeptide isrequired for forming a disulfide bridge in the second Kunitz-type domainwhich provides the conformation necessary for interaction with itsprotease substrate.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or Kunitz-type domaincontaining form of the protein generally will be retained when less thanthe majority of the residues of the complete or Kunitz-type domaincontaining form of the protein are removed from the N-terminus. Whethera particular polypeptide lacking N-terminal residues of a completeprotein retains such immunologic activities can readily be determined byroutine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the TFPI-3 shown in SEQ ID NO:2, up to the cysteine residueat position number 106, and polynucleotides encoding such polypeptides.In particular, the present invention provides polypeptides comprisingthe amino acid sequence of residues n-225 of SEQ ID NO:2, where n is aninteger in the range of 99 to 106. C106 is the position of the firstresidue from the N-terminus of the complete TFPI-3 polypeptide (shown inSEQ ID NO:2) believed to be required for protease inhibitory activity ofthe second Kuntiz-type domain of the complete TFPI-3 protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues of 99 to 225,100 to 225, 101 to 225, 102 to 225, 103 to 225, 104 to 225, 105 to 225and 106 to 225 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides also are provided.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. In the present case, since the protein of theinvention is a member of the TFPI polypeptide family, deletions ofC-terminal amino acids up to the cysteine at position 156 (C156) of SEQID NO:2 may retain some biological activity such asantithrombotic/protease inhibitor activity. Polypeptides having furtherC-terminal deletions including C156 of SEQ ID NO:2 would not be expectedto retain such biological activities because it is known that thisresidue in a TFPI-related polypeptide is required for forming adisulfide bridge in the second Kunitz-type domain which provides theconformation necessary for interaction with (and inhibition ofproteolytic and amidolytic activity of) factor Xa.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or Kunitz-type domaincontaining form of the protein generally will be retained when less thanthe majority of the residues of the complete or Kunitz-type domaincontaining form of the protein are removed from the C-terminus. Whethera particular polypeptide lacking C-terminal residues of a completeprotein retains such immunologic activities can readily be determined byroutine methods described herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of TFPI-3 shown in SEQ ID NO:2, up to the C156 of SEQ ID NO:2,and polynucleotides encoding such polypeptides. In particular, thepresent invention provides polypeptides having the amino acid sequenceof residues 99 to m of the amino acid sequence in SEQ ID NO:2, where mis any integer in the range of 156-225, and residue C156 is the positionof the first residue from the C-terminus of the complete TFPI-3polypeptide (shown in SEQ ID NO:2) believed to be required foramidolytic and proteolytic inhibitory activity of the second Kunitz-typedomain of the complete TFPI-3 protein.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues 99 to 156, 99 to157, 99 to 158, 99 to 159, 99 to 160, 99 to 161, 99 to 162, 99 to 163,99 to 164, 99 to 165, 99 to 166, 99 to 167, 99 to 168, 99 to 169, 99 to170, 99 to 171, 99 to 172, 99 to 173, 99 to 174, 99 to 175, 99 to 176,99 to 177, 99 to 178, 99 to 179, 99 to 180, 99 to 181, 99 to 182, 99 to183, 99 to 184, 99 to 185, 99 to 186, 99 to 187, 99 to 188, 99 to 189,99 to 190, 99 to 191, 99 to 192, 99 to 193, 99 to 194, 99 to 195, 99 to196, 99 to 197, 99 to 198, 99 to 199, 99 to 200, 99 to 201, 99 to 99 to203, 99 to 204, 99 to 205, 99 to 206, 99 to 207, 99 to 208, 99 to 209,99 to 210, 99 to 211, 99 to 212, 99 to 213, 99 to 214, 99 to 215, 99 to216, 99 to 217, 99 to 218, 99 to 219, 99 to 220, 99 to 221, 99 to 222,99 to 223, 99 to 224, and 99 to 225 of SEQ ID NO:2. Polynucleotidesencoding these polypeptides also are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n-m of SEQ ID NO:2, where n and mare integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete TFPI-3 amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97797, wherethis portion excludes from 126 to about 132 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97797; or from 1 to about 69 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97797.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the TFPI-3 polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the TFPI-3polypeptide which show substantial TFPI-3 polypeptide activity or whichinclude regions of TFPI-3 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions selected according to general rules known in theart so as have little effect on activity. For example, guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie, J. U. et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990), wherein the authors indicate that there are two main approachesfor studying the tolerance of an amino acid sequence to change. Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whicha Kunitz-type domain containing polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the above form of the polypeptide,such as an IgG Fc fusion region peptide or leader or secretory sequenceor a sequence which is employed for purification of the above form ofthe polypeptide or a proprotein sequence. Such fragments, derivativesand analogs are deemed to be within the scope of those skilled in theart from the teachings herein.

Thus, the TFPI-3 of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the TFPI-3 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vivo or in vitro proliferativeactivity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleveland et al., Crit.Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).

FIG. 2 shows an alignment of nine Kunitz-type domains. Preferred areTFPI-3 polypeptides having strongly conserved amino acids as shown inthe Kunitz consensus sequence SEQ ID NO:28. Where a TFPI-3 polypeptidehas an amino acid residue which is not identical to an amino acid at thesame position in the consensus sequence, the amino acid in the TFPI-3polypeptide mutein is preferably replaced with the amino acid shown inthe consensus sequence. Most highly preferred TFPI-3 muteins are thosehaving the amino acid sequence shown as the consensus sequence (SEQ IDNO:28). Polynucleotides encoding such polypeptides are also provided.Such polypeptides can easily be produced by method known to those ofskill in the are, for example, by solid phases synthesis methods.

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266-268 (1993) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

Since TFPI-3 is a member of the TFPI-related protein family, to modulaterather than completely eliminate biological activities of TFPI-3preferably mutations are made in sequences encoding amino acids in theTFPI-3 conserved Kunitz-type domains, i.e., in positions 11-61 and106-156 of SEQ ID NO:2, more preferably in residues within this regionwhich are not conserved in similar Kunitz-type domains among otherprotease inhibitors (see FIG. 3). More in particular, factor VIIa-tissuefactor exhibits a kinetic preference for synthetic substrates with a P1arginine residue (Kam, Ch. M. et al., Thromb. Haemostas., 64:133-137(1990)). Accordingly, replacement of the arginine at position 21 orposition 116 of SEQ ID NO:2 with any other amino acid results in aTFPI-3 mutant with conformational integrity but decreased activity;i.e., antagonists. Thus, forming part of the invention are polypeptidescomprising an amino acid sequence of the full-length TFPI-3, the matureTFPI-3 or a Kunitz-type domain containing form of TFPI-3 wherein theamino acid at position 21 and/or 116 of SEQ ID NO:2 is other thanarginine. Also forming part of the present invention are isolatedpolynucleotides comprising nucleic acid sequences which encode the aboveTFPI-3 mutants.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the TFPI-3 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-TFPI-3antibodies of the invention in methods which are well known in the artof protein purification.

The invention further provides an isolated TFPI-3 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of a consensus Kunitz-domain having the amino acidsequence shown as SEQ ID NO:28; and (b) the amino acid sequence of apolypeptide comprising the second Kunitz-type domain of TFPI-3 havingthe amino acid sequence at positions 106 to 156 in SEQ ID NO:2, or asencoded by the cDNA clone contained in ATCC Deposit NO. 97797; whereinthe polypeptide of (a) or (b) does not comprise a sequence shown as SEQID NO:29, SEQ ID NO:30, or SEQ ID NO:31.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TFPI-3polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the TFPI-3 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited cDNA clone can be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting TFPI-3 protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting TFPI-3 protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” TFPI-3 protein bindingproteins which are also candidate agonists and antagonists according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

Epitope-Bearing Portions

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) “Antibodies that react withpredetermined sites on proteins,” Science, 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Antigenic epitope-bearing peptides and polypeptidesof the invention are therefore useful to raise antibodies, includingmonoclonal antibodies, that bind specifically to a polypeptide of theinvention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate TFPI-3-specific antibodies include: apolypeptide comprising amino acid residues from about Asn-47 to aboutCys-61 in SEQ ID NO:2; a polypeptide comprising amino acid residues fromabout Asp-71 to about Thr-107; a polypeptide comprising amino acidresidues from about Glu-127 to about Asn-133; and a polypeptidecomprising amino acid residues from about Asn-142 to about Glu-150.These polypeptide fragments have been determined to bear antigenicepitopes of the TFPI-3 protein by the analysis of the Jameson-Wolfantigenic index, as shown in FIG. 3, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids.” Proc. Natl. Acad. Sci. USA 82:5131-5135; this“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol. 66:2347-2354 (1985). Immunogenic epitope-bearing peptides ofthe invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art. See, for instance, Geysen et al.,supra. Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C1-C7-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

Fusion Proteins

As one of skill in the art will appreciate, TFPI-3 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TFPI-3 protein orprotein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)).

Antibodies

TFPI-3-protein specific antibodies for use in the present invention canbe raised against the intact TFPI-3 protein or an antigenic polypeptidefragment thereof, which may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to TFPI-3 protein. Fab and F(ab′)2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the TFPI-3 protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of TFPI-3 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or TFPI-3 protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981) pp. 563-681). In general, such procedures involve immunizing ananimal (preferably a mouse) with a TFPI-3 protein antigen or, morepreferably, with a TFPI-3 protein-expressing cell. Suitable cells can berecognized by their capacity to bind anti-TFPI-3 protein antibody. Suchcells may be cultured in any suitable tissue culture medium; however, itis preferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP2O), available from the American TypeCulture Collection, Rockville, Md. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the TFPI-3 protein antigen.

Alternatively, additional antibodies capable of binding to the TFPI-3protein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, TFPI-3-protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the TFPI-3 protein-specific antibody can be blocked by theTFPI-3 protein antigen. Such antibodies comprise anti-idiotypicantibodies to the TFPI-3 protein-specific antibody and can be used toimmunize an animal to induce formation of further TFPI-3protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, TFPI-3protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of anti-TFPI-3 in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. See, for review, Morrison,Science 229:1202 (1985); Oi et al., Bio Techniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).

Hemostatic System-Related Disorders

Diagnosis

For a number of hemostatic system-related disorders, substantiallyaltered (increased or decreased) levels of TFPI-3 gene expression can bedetected in endothelial tissue or other cells or bodily fluids (e.g.,sera, plasma, urine, synovial fluid or spinal fluid) taken from anindividual having such a disorder, relative to a “standard” TFPI-3 geneexpression level, that is, the TFPI-3 expression level in endothelialtissue or bodily fluids from an individual not having the hemostaticsystem disorder. Thus, the invention provides a diagnostic method usefulduring diagnosis of a hemostatic disorder, which involves measuring theexpression level of the gene encoding the TFPI-3 protein in endothelialtissue or other cells or body fluid from an individual and comparing themeasured gene expression level with a standard TFPI-3 gene expressionlevel, whereby an increase or decrease in the gene expression levelcompared to the standard is indicative of an hemostatic system disorder.

Thus, the invention provides a diagnostic method useful during diagnosisof a hemostatic system disorder, which involves measuring the expressionlevel of the gene encoding the TFPI-3 protein in endothelial tissue orother cells or body fluid from an individual and comparing the measuredgene expression level with a standard TFPI-3 gene expression level,whereby an increase or decrease in the gene expression level compared tothe standard is indicative of a hemostatic system disorder.

Where a diagnosis of a disorder in the hemostatic system has alreadybeen made according to conventional methods, the present invention isuseful as a prognostic indicator, whereby patients exhibiting eitherenhanced or depressed TFPI-3 gene expression will experience a worseclinical outcome relative to patients expressing the gene at a levelnearer the standard level.

By “assaying the expression level of the gene encoding the TFPI-3protein” is intended qualitatively or quantitatively measuring orestimating the level of the TFPI-3 protein or the level of the mRNAencoding the TFPI-3 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level or mRNAlevel) or relatively (e.g., by comparing to the TFPI-3 protein level ormRNA level in a second biological sample). Preferably, the TFPI-3protein level or mRNA level in the first biological sample is measuredor estimated and compared to a standard TFPI-3 protein level or mRNAlevel, the standard being taken from a second biological sample obtainedfrom an individual not having the disorder or being determined byaveraging levels from a population of individuals not having a disorderof the hemostatic system. As will be appreciated in the art, once astandard TFPI-3 protein level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains TFPI-3 protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free TFPI-3 protein, endothelial tissue, andother tissue sources found to express complete or mature (! or“extracellular domain” of the TFPI-3 or a TFPI-3 receptor. Methods forobtaining tissue biopsies and body fluids from mammals are well known inthe art. Where the biological sample is to include mRNA, a tissue biopsyis the preferred source.

The present invention is useful for diagnosis or treatment of varioushemostatic system-related disorders in mammals, preferably humans. Suchdisorders include predisposition to vascular thrombosis, particularly inpost-stroke and post-cardiac surgery patients, hyperfibrinolytichemorrhage, traumatic-hemorrhagic shock, and hemophilia, and the like.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the TFPI-3 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Assaying TFPI-3 protein levels in a biological sample can occur usingantibody-based techniques. For example, TFPI-3 protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting TFPI-3 protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying TFPI-3 protein levels in a biological sampleobtained from an individual, TFPI-3 protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of TFPI-3protein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A TFPI-3 protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain TFPI-3 protein. Invivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Treatment

As noted above, TFPI-3 polynucleotides and polypeptides are useful fordiagnosis of conditions involving abnormally high or low expression ofTFPI-3 activities. Given the activities modulated by TFPI-3, it isreadily apparent that a substantially altered (increased or decreased)level of expression of TFPI-3 in an individual compared to the standardor “normal” level produces pathological conditions related to the bodilysystem(s) in which TFPI-3 is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since theTFPI-3 protein of the invention is a member of the TFPI-family themature form of the protein may be released in soluble form(s) from thecells which express TFPI-3 by proteolytic cleavage. Therefore, whensoluble TFPI-3 is added from an exogenous source to cells, tissues orthe body of an individual, the protein will exert its physiologicalactivities on its target cells of that individual.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of TFPI-3 activity in an individual,particularly disorders of the hemostasis system, can be treated byadministration of mature or Kunitz-type domain containing form of TFPI-3polypeptide (in a soluble form). Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofTFPI-3 activity comprising administering to such an individual apharmaceutical composition comprising an amount of an isolated TFPI-3polypeptide of the invention, effective to increase the TFPI-3 activitylevel in such an individual.

Alterations of the hemostatic system resulting in an increased incidenceof thrombotic disorders is a frequent consequence of neoplasia. Studiesin animal models with TFPI have demonstrated that TFPI infusionabrogates the disseminated intravascular coagulation triggered by TF andprevents arterial reocclusion following thrombolysis of clots induced byvessel injury. Intravascular coagulation is also known to be caused byendotoxin (Bronze, Jr., G. J., Seminars in Hematology, 29(3):159 (1992).

Therefore, TFPI-3 is useful in inhibiting intravascular clotting andpreventing the formation of fribrin clots both in vitro and in vivo. Thepolypeptides of the present invention are particularly useful foranticoagulant therapy in prophylaxis of venous thrombosis and astreatment for preventing its extension, as well as to provide low-doseregiment for prevention of postoperative deep venous thrombosis andpulmonary embolism in patients undergoing major abodominothoracicsurgery, particularly those who are at risk of developing thromboembolicdisease. TFPI-3 can also be used for the prophlaxis and treatment ofpulmonary embolism and atrial fibrillation with embolism. For example,pulmonary embolism represents the leading non-obstetric cause ofpost-partum death. Thus, TFPI-3 may be used to treat pregnant andpost-partum women. Additionally, TFPI-3 can be used to prevent clottingin arterial and heart surgery as well as for prevention of cerebralthrombosis in evolving stroke. TFPI-3 can be used both in treatingcoronary occlusion with acute myocardial infarction and in theprophylaxis and treatment of peripheral arterial emoblism. TFPI-3 mayalso be used in to treat sepsis, inflamatory diseases and transplantrejection. TFPI-3 can also be employed as an anticoagulant in bloodtransfusions, extracorporeal circulation, and dialysis procedures and inblood samples for laboratory purposes.

Similar to aprotinin, TFPI-3 polypeptides, particularly those containingonly one Kunitz-type domain including consensus Kunitz-type domainpolypeptides, may be particularly useful in the treatment ofhyperfilbronolytic hemorrhage and traumatic hemorrhagic shock as well asin diseases connected with excessive release of pancreatic elastase(pancreatitis), serum elastase (artherosclerosis), leukocyte elastase inacute and chronic inflammation with damage to connective tissue, indamage to vessel walls, in necrotic diseases, and degeneration of lungtissue.

Formulations

The TFPI-3 polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with TFPI-3 polypeptide alone), the site ofdelivery of the TFPI-3 polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of TFPI-3 polypeptide forpurposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofTFPI-3 polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the TFPI-3 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Pharmaceutical compositions containing the TFPI-3 of the invention maybe administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

The TFPI-3 polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release TFPI-3 polypeptide compositions also includeliposomally entrapped TFPI-3 polypeptide. Liposomes containing TFPI-3polypeptide are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTFPI-3 polypeptide therapy.

For parenteral administration, in one embodiment, the TFPI-3 polypeptideis formulated generally by mixing it at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the TFPI-3polypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The TFPI-3 polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of TFPI-3 polypeptide salts.

TFPI-3 polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic TFPI-3polypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

TFPI-3 polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-mil vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous TFPI-3 polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized TFPI-3 polypeptide usingbacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of TFPI-3 on cells, such as itsinteraction with TFPI-3-binding molecules such as receptor molecules. Anagonist is a compound which increases the natural biological functionsof TFPI-3 or which functions in a manner similar to TFPI-3, whileantagonists decrease or eliminate such functions.

In another aspect of this embodiment the invention provides a method foridentifying a receptor protein or other ligand-binding protein whichbinds specifically to a TFPI-3 polypeptide. For example, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds TFPI-3. Thepreparation is incubated with labeled TFPI-3 TFPI-3 and complexes ofTFPI-3 bound to the receptor or other binding protein are isolated andcharacterized according to routine methods known in the art.Alternatively, the TFPI-3 polypeptide may be bound to a solid support sothat binding molecules solubilized from cells are bound to the columnand then eluted and characterized according to routine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds TFPI-3, suchas a molecule of a signaling or regulatory pathway modulated by TFPI-3.The preparation is incubated with labeled TFPI-3 in the absence or thepresence of a candidate molecule which may be a TFPI-3 agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofTFPI-3 on binding the TFPI-3 binding molecule, are most likely to begood antagonists. Molecules that bind well and elicit effects that arethe same as or closely related to TFPI-3 are agonists.

TFPI-3-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofTFPI-3 or molecules that elicit the same effects as TFPI-3. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for TFPI-3 antagonists is a competitiveassay that combines TFPI-3 and a potential antagonist with a TFPI-3substrate under appropriate conditions for a competitive inhibitionassay. The substrate can be measured such that the activity of TFPI-3can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a receptor molecule, without inducingTFPI-3-induced activities, thereby preventing the action of TFPI-3 byexcluding TFPI-3 from binding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed, for example, in Okano, J. Neurochem. 56: 560 (1991);“Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.” CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooneyet al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA. For example, the 5′ coding portion of apolynucleotide that encodes the mature polypeptide of the presentinvention may be used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcriptionthereby preventing transcription and the production of TFPI-3. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into TFPI-3 polypeptide. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of TFPI-3 protein.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to promote coagulation inthe treatment of hemophilia. Antibodies against TFPI-3 may be employedto bind to and inhibit TFPI-3 activity to treat hemophilia. likewise,particularly preferred antagonists are TFPI-3 polypeptides containingonly a single Kunitz-type domain wherein the residue at the P1 positionis other than Arginine, as described above. Such polypeptides shouldbind to the same substrate as TFPI-3 but have reduced inhibitoryactivity. Any of the above antagonists may be employed in a compositionwith a pharmaceutically acceptable carrier, e.g., as hereinafterdescribed.

Gene Mapping

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a TFPI-3 protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1(a)

Expression and Purification of “His-tagged” TFPI Kunitz-type Domains-1and -2 in E. coli

The bacterial expression vector pQE9 (pD10) is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE9 encodes ampicillin antibiotic resistance (“Ampr”)and contains a bacterial origin of replication (“ori”), an IPTGinducible promoter, a ribosome binding site (“RBS”), six codons encodinghistidine residues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide.

The DNA sequence encoding the desired portion of the TFPI-3 proteincomprising the first Kunitz-type domains of the TFPI-3 amino acidsequence was amplified from the deposited cDNA clone using PCRoligonucleotide primers which anneal to the 5′ sequences of the desiredportion of the TFPI-3 cDNA and to sequences in the deposited construct3′ to the cDNA coding sequence. Additional nucleotides containingrestriction sites to facilitate cloning in the pQE9 vector were added tothe 5′ and 3′ primer sequences, respectively.

For cloning the first Kunitz-type domain of the TFPI-3 protein, the 5′primer had the sequence 5′ CGCAGATCTCGCAGCATCCACGACTTCTGCC 3′ (SEQ IDNO:20) containing the underlined BglII restriction site. The 3′ primerhad the sequence 5′ CGCAAGCTTTTAGGCATTCTCTGTGACAGTGGCA 3′ (SEQ ID NO:21)containing the underlined HindIII restriction site

For cloning the second Kunitz-type domain of the TFPI-3 protein, the 5′primer had the sequence 5′ CCCCGGATCCAGCGATATGTTCAACTATGAAGAATAC 3′ (SEQID NO:22) containing the underlined BamHI restriction site. The 3′primer had the sequence 5′ CCCCAAGCTTTTAATTCTCCTGCTGGCGGAAGCA 3′ (SEQ IDNO:23) containing the underlined HindIII restriction site.

One of ordinary skill in the art would appreciate, of course, that thepoint in the protein coding sequence where the primer anneals may bevaried to amplify a DNA segment encoding any desired portion of thecomplete TFPI-3 protein shorter or longer than the form of the proteindescribed here.

The amplified TFPI-3 DNA fragment and the vector pQE9 were digested withthe appropriate enzymes whose recognition sequence had been built intothe primers and the digested DNAs were then ligated together. Insertionof the TFPI-3 DNA into the restricted pQE9 vector places the TFPI-3protein coding region downstream from the IPTG-inducible promoter andin-frame with an initiating AUG and the six histidine codons.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),was used in carrying out the example described herein. This strain,which is only one of many that are suitable for expressing TFPI-3protein, is available commercially from QIAGEN, Inc., supra.Transformants were identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture was used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells weregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) was then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours. Cells then wereharvested by centrifugation.

The cells were then stirred for 3-4 hours at 4° C. in 6M guanidine-HCI,pH 8. The cell debris was removed by centrifugation, and the supernatantcontaining the TFPI-3 was loaded onto a nickel-nitrilo-tri-acetic acid(“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra).Proteins with a 6×His tag bind to the Ni-NTA resin with high affinityand can be purified in a simple one-step procedure (for details see: TheQIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatantwas loaded onto the column in 6 M guanidine-HCl, pH 8, the column wasfirst washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washedwith 10 volumes of 6 M guanidine-HCl pH 6, and finally the TFPI-3species were eluted with 6 M guanidine-HCl, pH 5 or pH 2.

The purified proteins were then renatured by dialyzing againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 5 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

Example 2

Cloning and Expression of TFPI-3 protein in a Baculovirus ExpressionSystem

In this example, the plasmid shuttle vector pA2 GP was used to insertthe cloned DNA encoding the Kunitz-type domains of the TFPI-3 protein,lacking its naturally associated secretory signal (leader) sequence,into a baculovirus for expression, using a baculovirus leader andstandard methods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa califonica nuclear polyhedrosis virus (AcMNPV) followed bythe secretory signal peptide (leader) of the baculovirus gp67 proteinand convenient restriction sites such as BamHI, Xba I and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpresses the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (19989).

The cDNA sequence encoding the Kunitz-type domains in the depositedclone, lacking the AUG initiation codon and the naturally associatedleader sequence shown in FIGS. 1A and 1B, were amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene. The 5′ primer had the sequence 5′CCCCAGATCTCGAACGCAGCATCCACGACTTCTGC 3′ (SEQ ID NO:24) containing theunderlined BglII restriction enzyme site followed by 24 nucleotides ofthe sequence of the mature TFPI-3 protein shown in SEQ ID NO:2,beginning with the indicated N-terminus of the Kunitz-type domaincontaining form of the TFPI-3 protein. The 3′ primer had the sequence 5CCCCTCTAGATTAATTCTCCTGCTGGCGGAAGCAGC 3′ (SEQ ID NO:25) containing theunderlined XbaI restriction site followed by an artificial complimentarystop codon, TTA, and 23 nucleotides complementary to the 3′ codingsequence in FIGS. 1A and 1B. The resulting fragment encodes seven TFPI-3amino acids amino to the first Kunitz-type domain and six amino acidscarboxy terminal to the second Kunitz-type domain shown in FIGS. 1A and1B.

The amplified fragment was isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then was digested with BglII and XbaI and again ispurified on a 1% agarose gel.

The plasmid was digested with the restriction enzymes BglII and XbaI anddephosphorylated using calf intestinal phosphatase, using routineprocedures known in the art. The DNA was then isolated from a 1% agarosegel using a commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.).

Fragment and the dephosphorylated plasmid were ligated together with T4DNA ligase. E. coli HB101 and XL-1 Blue (Statagene Cloning Systems, LaJolla, Calif.) cells were transformed with the ligation mixture andspread on culture plates. Bacteria were identified that contain theplasmid with the human TFPI-3 gene by digesting DNA from individualcolonies using BglII and XbaI and then analyzing the digestion productby gel electrophoresis. The sequence of the cloned fragment wasconfirmed by DNA sequencing. This plasmid is designated hereinpA2TFPI-3.

Five μg of the plasmid pA2TFPI-3 was co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BaculoGold™ virus DNA and 5 μg of theplasmid pA2TFPI-3 were mixed in a sterile well of a microtiter platecontaining 50 μg of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μg Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate was then incubated for 5hours at 27° C. The transfection solution was then removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum was added. Cultivation was then continued at 27° C. for four days.

After four days the supernatant was collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) was used to allow easyidentification and isolation of gal-expressing clones, which producedblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10). After appropriate incubation, blue stained plaques werepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses was then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus was used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then they are stored at 4° C. Therecombinant virus is called V-TFPI-3.

To verify the expression of the TFPI-3 gene Sf9 cells were grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cellswere infected with the recombinant baculovirus V-TFPI-3 at amultiplicity of infection (“MOI”) of about 2. The proteins in thesupernatant as well as the intracellular proteins were analyzed bySDS-PAGE.

Example 3

Cloning and Expression of TFPI-3 in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a)

Cloning and Expression in COS Cells

The expression plasmid in this illustrative example, pTFPI-3HA, is madeby cloning the cDNA encoding the complete TFPI-3 protein into theexpression vector pcDNAI/Amp or pcDNAIII (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37: 767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the complete TFPI-3 polypeptide is cloned intothe polylinker region of the vector so that recombinant proteinexpression is directed by the CMV promoter. The plasmid constructionstrategy is as follows. The TFPI-3 cDNA of the deposited clone isamplified using primers that contain convenient restriction sites, muchas described above for construction of vectors for expression of TFPI-3in E. coli. Suitable primers include the following, which are used inthis example. The 5′ primer, containing the underlined BglII site, aKozak sequence, an AUG start codon, and 5′ coding region of the completeTFPI-3 polypeptide, has the following sequence: 5′CCCCAGATCTGCCATCATGGCGCAGCTGTGCGGGCTGA 3′ (SEQ ID NO:26). The 3′ primer,containing the underlined XbaI restriction site has the followingsequence: 5′ CGCTCTAGATCACAGGACATATGTGTTCTT 3′ (SEQ ID NO:27).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BglII and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding the completeTFPI-3 polypeptide

For expression of recombinant TFPI-3, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of TFPI-3 by thevector.

Expression of the TFPI-3-HA fusion protein is detected by radiolabelingand immunoprecipitation, using methods described in, for example Harlowet al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. citedabove. Proteins are precipitated from the cell lysate and from theculture media using an HA-specific monoclonal antibody. The precipitatedproteins then are analyzed by SDS-PAGE and autoradiography. Anexpression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b)

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of TFPI-3 polypeptide. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the TFPI-3 polypeptide in a regulated wayin mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89:5547-5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes BglII and XbaIand then dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete TFPI-3 polypeptide is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the desired portion of the gene. The 5′ primer containingthe underlined BglII site, a Kozak sequence, and an AUG start codon, hasthe following sequence: 5′ CCCCAGATCTGCCATCATGGCGCAGCTGTGCGGGCTGA 3′(SEQ ID NO:26). The 3′ primer, containing the underlined XbaIrestriction site, has the following sequence: 5′CGCTCTAGATCACAGGACATATGTGTTCTT 3′ (SEQ ID NO:27).

The amplified fragment is digested with the endonucleases BglII and XbaIand then purified again on a 1% agarose gel. The isolated fragment andthe dephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4

Tissue Distribution of TFPI-3 mRNA Expression

Northern blot analysis was carried out to examine TFPI-3 gene expressionin human tissues, including spleen, thymus, small intestine, colon,peripheral blood leukocytes, prostate and testis, using methodsdescribed by, among others, Sambrook et al., cited above. A cDNA probecontaining the entire nucleotide sequence of the TFPI-3 protein (SEQ IDNO:1) was labeled with ³²P using the rediprime™ DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe was purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT1200-1. The purified labeled probe was then used to examinevarious human tissues for TFPI-3 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) were obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

Expression of TFPI-3 was observed in all tissues tested, but was highestin prostate and testis.

Example 5

TFPI-3 Trypsin Inhibition Assay

The expression, purification, and renaturation of the first Kunitz-typedomain of the TFPI-3 protein (TFPI-3-1) in E. coli is described inExample 1. The refolded samples were centrifuged at 15,000 g for 10minutes to remove insoluble material. The purified protein was found tobe more than 90% pure as observed by SDS-PAGE. The concentration of theprotein was 1 mg/ml as measured by the Bradford assay.

The assay described in this example involves the use ofp-toluenesulphonyl-L-arginine methyl ester (TAME) as a substrate fortrypsin and was first described by Hummel, B. C. W. (Can J. Biochem.Physil., 37:1393 (1959)). Lyophilized aliquots of TAME (0.001 M TAME and0.01 M calcium in 0.04 M Tris buffer, pH7.8 to 8.2, when reconstitutedaccording to manufacturer's suggested protocol) were purchased fromWorthington Biochemical Corporation (Freehold, N.J.). Trypsin (Sigma)was prepared in 50 mM Tris buffer, pH 8.0, at a concentration of 10mg/ml. Ten ml trypsin was added to acetate buffer (50 mM Na-acetate,pH5.0, 200 mM NaCl) containing 1.25 ug, 2.50 ug, 5.0 ug, 10.0 ug and20.0 ug of purified first Kunitz domain, in total volume of 100 ml, andincubated for 10 minutes at room temperature. After incubation, thesamples were added to 1 ml of TAME reagent and absorbance at 247 nm wasmonitored at 37° C. in a period of 30 min according to Worthington'sprotocol. His-tagged Cripto (a TGF-a family member), purified in thesame way as TFPI-3-1, was used as a negative control.

Results

The results are shown in FIG. 4. As can be seen, TFPI-3-1 (first Kunitztype domain of TFPI-3) did not appear to inhibit trypsin activity at1.25 and 2.50 mg doses. When 5.00 mg of TFPI-3-1 was included,approximately 60% of trypsin activity was inhibited. At 10.00 mg,trypsin activity was reduced to 13% as compared to the buffer-onlycontrol. Trypsin activity was almost totally abolished at 20.00 mg ofTFPI-3-1. When the same concentrations of Cripto were used as a controlno trypsin inhibiting activity could be observed.

The trypsin inhibiting activity of TFPI-3-1 was further confirmed byassaying duplicate samples in a single experiment. Trypsin (100 ng) wasincubated with buffer only, 10 mg of Cripto or 10 mg of TFPI-3-1. Afterthe addition of TAME substrate and absorbance at 247 nm was measured.Approximately 7% of trypsin activity was left in the TFPI-3-1 samples ascompared to the buffer only samples (Table 2, below).

TABLE 2 Effects of TFPI-3-1 and Cripto control on inhibition of trypsinactivity. Buffer only TFPI-3-1 (10 μg) Cripto Control (10 μg) 85 ± 3.5 6± 8.5 105 ± 0.7 Numbers denote Trypsin Activity (10⁻⁴ * ΔA/min), n = 2.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

31 1610 base pairs nucleic acid single linear DNA (genomic) not providedCDS 361..1116 sig_peptide 361..439 mat_peptide 442..1116 1 CTGCCCGGCCACCTTCGGGA GCCGCTTCCA ATAGGCGTTC GCCATTGGCT CTGGCGACCT 60 CCGCGCGTTGGGAGGTGTAG CGCGGCTCTG AACGCGCTGA GGGCCGTTGA GTGTCGCAGG 120 CGGCGAGGGCGCGAGTGAGG AGCAGACCCA GGCATCGCGC GCCGAGAAGG CCGGGCGTCC 180 CCACACTGAAGGTCCGGAAA GGCGACTTCC GGGGGCTTTG GCACCTGGCG GACCCTCCCG 240 GAGCGTCGGCACCTGAACGC GAGGCGCTCC ATTGCGCGTG CGCGTTGAGG GGCTTCCCGC 300 ACCTGATCGCGAGACCCCAA CGGCTGGTGG CGTCGCCTGC GCGTCTCGGC TGAGCTGGCC 360 ATG GCG CAGCTG TGC GGG CTG AGG CGG AGC CGG GCG TTT CTC GCC CTG 408 Met Ala Gln LeuCys Gly Leu Arg Arg Ser Arg Ala Phe Leu Ala Leu -27 -25 -20 -15 CTG GGATCG CTG CTC CTC TCT GGG GTC CTG GCG GCC GAC CGA GAA CGC 456 Leu Gly SerLeu Leu Leu Ser Gly Val Leu Ala Ala Asp Arg Glu Arg -10 -5 1 5 AGC ATCCAC GAC TTC TGC CTG GTG TCG AAG GTG GTG GGC AGA TGC CGG 504 Ser Ile HisAsp Phe Cys Leu Val Ser Lys Val Val Gly Arg Cys Arg 10 15 20 GCC TCC ATGCCT AGG TGG TGG TAC AAT GTC ACT GAC GGA TCC TGC CAG 552 Ala Ser Met ProArg Trp Trp Tyr Asn Val Thr Asp Gly Ser Cys Gln 25 30 35 CTG TTT GTG TATGGG GGC TGT GAC GGA AAC AGC AAT AAT TAC CTG ACC 600 Leu Phe Val Tyr GlyGly Cys Asp Gly Asn Ser Asn Asn Tyr Leu Thr 40 45 50 AAG GAG GAG TGC CTCAAG AAA TGT GCC ACT GTC ACA GAG AAT GCC ACG 648 Lys Glu Glu Cys Leu LysLys Cys Ala Thr Val Thr Glu Asn Ala Thr 55 60 65 GGT GAC CTG GCC ACC AGCAGG AAT GCA GCG GAT TCC TCT GTC CCA AGT 696 Gly Asp Leu Ala Thr Ser ArgAsn Ala Ala Asp Ser Ser Val Pro Ser 70 75 80 85 GCT CCC AGA AGG CAG GATTCT GAA GAC CAC TCC AGC GAT ATG TTC AAC 744 Ala Pro Arg Arg Gln Asp SerGlu Asp His Ser Ser Asp Met Phe Asn 90 95 100 TAT GAA GAA TAC TGC ACCGCC AAC GCA GTC ACT GGG CCT TGC CGT GCA 792 Tyr Glu Glu Tyr Cys Thr AlaAsn Ala Val Thr Gly Pro Cys Arg Ala 105 110 115 TCC TTC CCA CGC TGG TACTTT GAC GTG GAG AGG AAC TCC TGC AAT AAC 840 Ser Phe Pro Arg Trp Tyr PheAsp Val Glu Arg Asn Ser Cys Asn Asn 120 125 130 TTC ATC TAT GGA GGC TGCCGG GGC AAT AAG AAC AGC TAC CGC TCT GAG 888 Phe Ile Tyr Gly Gly Cys ArgGly Asn Lys Asn Ser Tyr Arg Ser Glu 135 140 145 GAG GCC TGC ATG CTC CGCTGC TTC CGC CAG CAG GAG AAT CCT CCC CTG 936 Glu Ala Cys Met Leu Arg CysPhe Arg Gln Gln Glu Asn Pro Pro Leu 150 155 160 165 CCC CTT GGC TCA AAGGTG GTG GTT CTG GCG GGG CTG TTC GTG ATG GTG 984 Pro Leu Gly Ser Lys ValVal Val Leu Ala Gly Leu Phe Val Met Val 170 175 180 TTG ATC CTC TTC CTGGGA GCC TCC ATG GTC TAC CTG ATC CGG GTG GCA 1032 Leu Ile Leu Phe Leu GlyAla Ser Met Val Tyr Leu Ile Arg Val Ala 185 190 195 CGG AGG AAC CAG GAGCGT GCC CTG CGC ACC GTC TGG AGC TCC GGA GAT 1080 Arg Arg Asn Gln Glu ArgAla Leu Arg Thr Val Trp Ser Ser Gly Asp 200 205 210 GAC AAG GAG CAG CTGGTG AAG AAC ACA TAT GTC CTG TGACCGCCCT 1126 Asp Lys Glu Gln Leu Val LysAsn Thr Tyr Val Leu 215 220 225 GTCGCCAAGA GGACTGGGGA AGGGAGGGGAGACTATGTGT GAGCTTTTTT TAAATAGAGG 1186 GATTGACTCG GATTTGAGTG ATCATTAGGGCTGAGGTCTG TTTCTCTGGG AGGTAGGACG 1246 GCTGCTTCCT GGTCTGGCAG GGATGGGTTTGCTTTGGAAA TCCTCTAGGA GGCTCCTCCT 1306 CGCATGGCCT GCAGTCTGGC AGCAGCCCCGAGTTGTTTCC TCGCTGATCG ATTTCTTTCC 1366 TCCAGGTAGA GTTTTCTTTG CTTATGTTGAATTCCATTGC CTCTTTTCTC ATCACAGAAG 1426 TGATGTTGGA ATCGTTTCTT TTGTTTGTCTGATTTATGGT TTTTTTAAGT ATAAACAAAA 1486 GTTTTTTATT AGCATTCTGA AAGAAGGAAAGTAAAATGTA CAAGTTTAAT AAAAAGGGGC 1546 CTTCCCCTTT AGAATAAATT TCAGCATGTGCTTTCAAAAA AAAAAAAAAA AAAAAAAAAA 1606 AAAA 1610 252 amino acids aminoacid linear protein not provided 2 Met Ala Gln Leu Cys Gly Leu Arg ArgSer Arg Ala Phe Leu Ala Leu -27 -25 -20 -15 Leu Gly Ser Leu Leu Leu SerGly Val Leu Ala Ala Asp Arg Glu Arg -10 -5 1 5 Ser Ile His Asp Phe CysLeu Val Ser Lys Val Val Gly Arg Cys Arg 10 15 20 Ala Ser Met Pro Arg TrpTrp Tyr Asn Val Thr Asp Gly Ser Cys Gln 25 30 35 Leu Phe Val Tyr Gly GlyCys Asp Gly Asn Ser Asn Asn Tyr Leu Thr 40 45 50 Lys Glu Glu Cys Leu LysLys Cys Ala Thr Val Thr Glu Asn Ala Thr 55 60 65 Gly Asp Leu Ala Thr SerArg Asn Ala Ala Asp Ser Ser Val Pro Ser 70 75 80 85 Ala Pro Arg Arg GlnAsp Ser Glu Asp His Ser Ser Asp Met Phe Asn 90 95 100 Tyr Glu Glu TyrCys Thr Ala Asn Ala Val Thr Gly Pro Cys Arg Ala 105 110 115 Ser Phe ProArg Trp Tyr Phe Asp Val Glu Arg Asn Ser Cys Asn Asn 120 125 130 Phe IleTyr Gly Gly Cys Arg Gly Asn Lys Asn Ser Tyr Arg Ser Glu 135 140 145 GluAla Cys Met Leu Arg Cys Phe Arg Gln Gln Glu Asn Pro Pro Leu 150 155 160165 Pro Leu Gly Ser Lys Val Val Val Leu Ala Gly Leu Phe Val Met Val 170175 180 Leu Ile Leu Phe Leu Gly Ala Ser Met Val Tyr Leu Ile Arg Val Ala185 190 195 Arg Arg Asn Gln Glu Arg Ala Leu Arg Thr Val Trp Ser Ser GlyAsp 200 205 210 Asp Lys Glu Gln Leu Val Lys Asn Thr Tyr Val Leu 215 220225 59 amino acids amino acid single linear protein not provided 3 ArgArg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys 1 5 10 15Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln 20 25 30Thr Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser 35 40 45Ala Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 51 amino acids aminoacid single linear protein not provided 4 Cys Ala Phe Lys Ala Asp AspGly Pro Cys Lys Ala Ile Met Lys Arg 1 5 10 15 Phe Phe Phe Asn Ile PheThr His Gln Cys Glu Glu Phe Ile Tyr Gly 20 25 30 Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys 35 40 45 Lys Met Cys 50 51 aminoacids amino acid single linear protein not provided 5 Cys Phe Leu GluGlu Asp Pro Gly Ile Cys His Gly Tyr Ile Thr Arg 1 5 10 15 Tyr Phe TyrAsn Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly 20 25 30 Gly Cys LeuGly Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys 35 40 45 Asn Ile Cys50 51 amino acids amino acid single linear protein not provided 6 CysLeu Thr Pro Ala Asp Arg Gly Leu Cys Arg Ala Asn Glu Asn Arg 1 5 10 15Phe Tyr Tyr Asn Ser Val Ile Gly Lys Cys Arg Pro Phe Lys Tyr Ser 20 25 30Gly Cys Gly Gly Asn Glu Asn Asn Phe Thr Ser Lys Gln Glu Cys Leu 35 40 45Arg Ala Cys 50 51 amino acids amino acid single linear protein notprovided 7 Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala Leu Leu LeuArg 1 5 10 15 Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln Phe LeuTyr Gly 20 25 30 Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr Thr Trp Glu AlaCys Asp 35 40 45 Asp Ala Cys 50 54 amino acids amino acid single linearprotein not provided 8 Cys Arg Leu Gln Val Ser Val Asp Asp Gln Cys GluGly Ser Thr Glu 1 5 10 15 Lys Tyr Phe Phe Asn Leu Ser Ser Met Thr CysGlu Lys Phe Phe Ser 20 25 30 Gly Gly Cys His Arg Asn Arg Ile Glu Asn ArgPhe Pro Asp Glu Ala 35 40 45 Thr Cys Met Gly Phe Cys 50 51 amino acidsamino acid single linear protein not provided 9 Cys Tyr Ser Pro Lys AspGlu Gly Leu Cys Ser Ala Asn Val Thr Arg 1 5 10 15 Tyr Tyr Phe Asn ProArg Tyr Arg Thr Cys Asp Ala Phe Thr Tyr Thr 20 25 30 Gly Cys Gly Gly AsnAsp Asn Asn Phe Val Ser Arg Glu Asp Cys Lys 35 40 45 Arg Ala Cys 50 51amino acids amino acid single linear protein not provided 10 Cys Leu ValSer Lys Val Val Gly Arg Cys Arg Ala Ser Met Pro Arg 1 5 10 15 Trp TrpTyr Asn Val Thr Asp Gly Ser Cys Gln Leu Phe Val Tyr Gly 20 25 30 Gly CysAsp Gly Asn Ser Asn Asn Tyr Leu Thr Lys Glu Glu Cys Leu 35 40 45 Lys LysCys 50 51 amino acids amino acid single linear protein not provided 11Cys Thr Ala Asn Ala Val Thr Gly Pro Cys Arg Ala Ser Phe Pro Arg 1 5 1015 Trp Tyr Phe Asp Val Glu Arg Asn Ser Cys Asn Asn Phe Ile Tyr Gly 20 2530 Gly Cys Arg Gly Asn Lys Asn Ser Tyr Arg Ser Glu Glu Ala Cys Met 35 4045 Leu Arg Cys 50 499 base pairs nucleic acid single linear DNA(genomic) not provided 12 CGAGGGCGCG AGTAAGGAGC AGACCCAGGC ATCGCGCGCCGAGAAGGCCG GGCGTCCCCA 60 CACTGAAGGT CCGGAAAGGC GACTTCCGGG GGCTTTGGCACCTGGCGGAC CCTCCCGGAG 120 CGTCGGCACC TGAACGCGAG GCGCTCCATT GCGCGTGCGNTTTGAGGGGC TTCCCGCACC 180 TGATCGCGAG ACCCCAACGG CTGGTGGCGT CGCTGCGCGTCTNGGCTGAG CTGGCCATGG 240 CGCAGTGTTG CGGGCTTGAG GCGGACGCNG CGTTTNTNGCCTGCTNGGAT CGCTGCTTCT 300 CTCTGGGGTC CTNGCGGCCG ACCGAGAACG NAGNATCNANGATTTTTNCN TGGTGTCGAA 360 GTTGGTGGGA AATTCCGGGC TTCANTNCTA AGTGNTGTAAATTTAATTAC GGTCCTNCAA 420 TNTTTTTTAN TNGGGTTTAC GGAAAGAATA ATNACTTNCAAGAGNTTCTT AANAATTTCA 480 TTAAAANNAT CAAGGTACT 499 287 base pairsnucleic acid single linear DNA (genomic) not provided 13 AGTCACTGGGCCTTGCCGTG CATCCTTCCC ACGCTGGTAC TTTGACGTGG AGAGGAACTC 60 CTGCAATAACTTCATCTATG GAGGCTGCCG GGGCAATAAG AACAGCTACC GCTCTGAGGA 120 GGCCTGCATGCTCCGCTGCT TCCGCCAGCA GGAGAATCCT CCCCTGCCCC TTGGCTCAAA 180 GNTGGTGGTTCTNGCGGGGC TGTTCGTGAT GGTGTTGATC CTCTTCCTNG GAGCCTCCAT 240 GGTCTACCTTATCCGGGTGG CACGGAGGAA CCAGGAGCGT GCCCTGC 287 273 base pairs nucleic acidsingle linear DNA (genomic) not provided 14 TTTTTTTTAT TCTAAAGGGGAAGGCCCCTT TNTATTAAAC TTGTACATTT TACTTTCNTT 60 CTTTCAGAAT NNNAATAAAAAACTTTTGTT TATACTTAAA AAAACCATAA ATCAGACAAA 120 CAAAAGAAAC GATTCCAACATCACTTCTGT GATGAGAAAA GAGGCAATGG AATTCAACAT 180 AAGCAAAGAA AACTNTACCTGGNGGAAAGA AATCGATCAG CGAGGAAACA ACTCGGGGCT 240 GCTGCCAGAC TNCAGGCCATGCGAGGAGGA GCC 273 256 base pairs nucleic acid single linear DNA(genomic) not provided 15 TTTTTTTTAT TCTAAAGGGG AAGGCCCCTT TTTATTAAACTTGTACATTT TACTTTCCTT 60 CTTTCAGAAT GCTAATAAAA AACTTTTGTT TATACTTAAAAAAACCATAA ATCAGACAAA 120 CAAAAGAAAC GATTCCAACA TCACTTCTGT GATGAGAAAAGAGGCAATGG AATTCAACAT 180 AAGCAAAGAA AACTCTACCT GGNGGAAAGA AATCGATCAGCGAGGAAACA ACTCGGGGCT 240 GCTGCCAGAC TGCAGG 256 496 base pairs nucleicacid single linear DNA (genomic) not provided 16 AATTCGGCAC GAGGTACAATNTCACTNACG GATCCTGCCA GCTGTTTGTN TATGGGGGCT 60 GTNACGGAAA CAGCAATAATTACCTGACCA AGGAGGAGTG CCTCAAGAAA TGTGCCACTG 120 TCACAGAGAA TGCCACGGGTGNCCTGGCCA CCAGCAGGAN TGCAGCGGAT TCCTCTGTCC 180 CAAGTGCTCC CAGAAGGCAGGATTNTNAAG ACCACTCCAG CGGTATGTTC AACTATNGAG 240 GATACTTGCA CCGNCAACGGAGTTCACTAG GGCTTTGCCG TGCATCCTTT TCCCACGGTT 300 GGTACTTTTN AACGGNGGAGGAGGAACTTC CTNNNAATAA ANTTNATNTT NTGGGNGGTN 360 NTCCGGGGGN ATNAAGNANCAATNACCGNT TTTAANGGNG GNNTTNANTT NTCNNNTTTT 420 TTTCCCCNNA NGGGGNTTNTNCCNNTNCCN TNGGTANAAA GGGGGGNTTT TTNGGGGATT 480 TTTTGGAAAA TNAAAT 496201 base pairs nucleic acid single linear DNA (genomic) not provided 17GAGGAACCAG GAGCGTGCCC TNCGCACCGT CTGGAGCTCC GGAGATGACA AGGAGCAGCT 60GGTGAAGAAC ACATATGTCC TGTGACCGCC CTGTCGCCAA GAGGACTNGG GAAGGGAGGG 120GAGACTATGT GTGAGCTTTT TTTAAATAGA GGGATTGACT CGGATTTGAG TGATCATTAG 180GGCTGAGGTC TGTTTCTCTG G 201 419 base pairs nucleic acid single linearDNA (genomic) not provided 18 GGCAGAGGTC TTCCCGCACC TAAATCGCAAGACCCCAACG GCTGGTGGCG TCGCTGNCCC 60 GTNTCGGCTG GGCTGGCCAT GGCGCATGTGCCGGGCCTGA GGCGGACCGN CGTTTNTNGC 120 CTGCTGGGAA TCGCTGCTCC TTTNTGGGGTCCTGGCGGCC GACCGAAAAC GCCNGCATCC 180 ACNANTTCTG CCTGGTGTCG AAGGTGGTGGGCAGATGCCG GGCCTCCATG CCTAGGTGGT 240 GGTACAATGT TNACTAACGG ATCCTGGCCAGTTTTTGTGT ATGGGGGGCT TTTAACGGGA 300 AACAGCAATA ATTTAACTNG ACCAAGGAGGAGTTGCCTTC AAGAAATGTT GNCCATNTTT 360 NAAAAGGGNA TNCCCAGGGG TTAACCTGGGCCACCANAGG GATTTAAGGG GTTTTNTTT 419 437 base pairs nucleic acid singlelinear DNA (genomic) not provided 19 GAATTCGGCA GAGCCCGTGA CTTCCNCCAGCAGGAGAATC CTCCCCTGCC CCTTGGCTCA 60 AAGGCCCTGG AAGCCCACCT CCNAGAAGCCCGGTGTGGGG GCGGGCCACG GGGGAGAATC 120 CCAAGCTCAG TCCCCACAAA GTTCAGGGCCGGTCGGAGGC AGGGGCAGGT CCGGGTCCAA 180 AGCAAGGACA CCACAGCTCT TCCGAACTCCAGCAGCAGCT TCCAGCNATT TCGGAACACG 240 GATGTAAAGT TCCCACGNCT TGCTGGCTTCCAAGCACNAC GGAGAAGNCA TTCCCCGGGG 300 CAAGGNCCAA AGNAAGGCCC CAAAAGTTGAAGGAAGAAGG GAGGAAGGGG CAAGNNAGGN 360 GGAAGGGGCA AGAAGGAAGG AGGTTTCCCCCATTTGNNAG GGGNCTTNGN ACAGGTTTCC 420 ATTTAAAACC TTTNCTT 437 31 basepairs nucleic acid single linear DNA (genomic) not provided 20CGCAGATCTC GCAGCATCCA CGACTTCTGC C 31 34 base pairs nucleic acid singlelinear DNA (genomic) not provided 21 CGCAAGCTTT TAGGCATTCT CTGTGACAGTGGCA 34 37 base pairs nucleic acid single linear DNA (genomic) notprovided 22 CCCCGGATCC AGCGATATGT TCAACTATGA AGAATAC 37 34 base pairsnucleic acid single linear DNA (genomic) not provided 23 CCCCAAGCTTTTAATTCTCC TGCTGGCGGA AGCA 34 35 base pairs nucleic acid single linearDNA (genomic) not provided 24 CCCCAGATCT CGAACGCAGC ATCCACGACT TCTGC 3536 base pairs nucleic acid single linear DNA (genomic) not provided 25CCCCTCTAGA TTAATTCTCC TGCTGGCGGA AGCAGC 36 38 base pairs nucleic acidsingle linear DNA (genomic) not provided 26 CCCCAGATCT GCCATCATGGCGCAGCTGTG CGGGCTGA 38 30 base pairs nucleic acid single linear DNA(genomic) not provided 27 CGCTCTAGAT CACAGGACAT ATGTGTTCTT 30 51 aminoacids amino acid single linear protein not provided 28 Cys Leu Leu ProAla Asp Thr Gly Pro Cys Arg Ala Ser Ile Thr Arg 1 5 10 15 Tyr Phe TyrAsn Val Xaa Thr Gly Ser Cys Glu Xaa Phe Val Tyr Gly 20 25 30 Gly Cys GlyGly Asn Arg Asn Asn Phe Glu Ser Leu Glu Glu Cys Lys 35 40 45 Arg Ala Cys50 15 amino acids amino acid single Not Relevant peptide not provided 29Ala Asp Arg Glu Arg Ser Ile His Asp Phe Xaa Leu Val Ser Lys 1 5 10 15 29amino acids amino acid single Not Relevant peptide not provided 30 LysVal Val Gly Arg Xaa Arg Ala Ser Met Pro Arg Trp Trp Tyr Asn 1 5 10 15Val Thr Asp Gly Ser Xaa Gln Leu Phe Val Tyr Gly Gly 20 25 26 amino acidsamino acid single Not Relevant peptide not provided 31 Ala Thr Val ThrGlu Asn Ala Thr Gly Asp Leu Ala Thr Ser Arg Asn 1 5 10 15 Ala Ala AspSer Ser Val Pro Ser Ala Pro 20 25

What is claimed is:
 1. An isolated polypeptide comprising amino acidshaving a sequence which is at least 90% identical to referencepolypeptide sequence SEQ ID NO:28, wherein said polypeptide has proteaseinhibitor activity.
 2. The polypeptide of claim 1, comprising aminoacids having a sequence which is at least 95% identical to referencepolypeptide sequence SEQ ID NO:28.
 3. The polypeptide of claim 2, whichcomprises SEQ ID NO:28.
 4. The polypeptide of claim 1, furthercomprising a heterologous polypeptide.
 5. The polypeptide of claim 4,wherein said heterologous polypeptide is an immunoglobulin constantregion.
 6. A composition comprising the polypeptide of claim 1 and acarrier.
 7. The polypeptide of claim 1, which inhibits the proteaseactivity of trypsin.
 8. The polypeptide of claim 1, which inhibits theamidolytic activity of factor VIIa-tissue factor.
 9. An isolatedpolypeptide comprising at least 9 contiguous amino acids of SEQ IDNO:28; wherein said polypeptide binds an antibody with specificity forapolypeptide consisting of SEQ ID NO:28.
 10. The polypeptide of claim 9,comprising at least 15 contiguous amino acids of SEQ ID NO:28.
 11. Thepolypeptide of claim 10, comprising at least 30 contiguous amino acidsof SEQ ID NO:28.
 12. The polypeptide of claim 9, further comprising aheterologous polypeptide.
 13. The polypeptide of claim 12, wherein saidheterologous polypeptide is an immunoglobulin constant region.
 14. Acomposition comprising the polypeptide of claim 9 and a carrier.
 15. Amethod of using the polypeptide of claim 9 produce an antibody,comprising administering said polypeptide to an animal, and recoveringsaid antibody from said animal.
 16. An isolated polypeptide comprisingamino acids having a sequence which, except for one amino acidsubstitution at position Arg-116, is identical to amino acids 106 to 156of SEQ ID NO:2.
 17. The isolated polypeptide of claim 16, wherein saidpolypeptide has protease inhibitor activity; and wherein said proteaseinhibitor activity is reduced relative to that of a referencepolypeptide comprising amino acids 106 to 156 of SEQ ID NO:2.
 18. Thepolypeptide if claim 16, further comprising a heterologous polypeptide.19. The polypeptide of claim 18, wherein said heterologous polypeptideis an immunoglobulin constant region.
 20. A composition comprising thepolypeptide of claim 16 and a carrier.
 21. An isolated polypeptidefragment consisting of amino acids whose sequence is at least 90%identical to amino acids 106 to 156 of SEQ ID NO:2; wherein saidpolypeptide fragment has protease inhibitor activity.
 22. Thepolypeptide fragment of claim 21, consisting of amino acids whosesequence is at least 95% identical to amino acids 106 to 156 of SEQ IDNO:2.
 23. The polypeptide fragment of claim 22, consisting of aminoacids 106 to 156 of SEQ ID NO:2.
 24. An isolated polypeptide consistingof the polypeptide fragment of claim 21, and a heterologous polypeptidefused to said polypeptide fragment.
 25. The polypeptide of claim 24,wherein said heterologous polypeptide comprises an immunoglobulinconstant region.
 26. A composition comprising the polypeptide fragmentof claim 21 and a carrier.
 27. The polypeptide fragment of claim 21,which inhibits the activity of trypsin.
 28. The polypeptide fragment ofclaim 21, which inhibits the amidolytic activity of factor VIIa-tissuefactor.
 29. An isolated polypeptide fragment consisting of amino acidshaving a sequence which, except for one amino acid substitution atposition Arg-116, is identical to amino acids 106 to 156 of SEQ ID NO:2;wherein said polypeptide fragment has protease inhibitor activity; andwherein said protease inhibitor activity is reduced relative to that ofa reference polypeptide fragment consisting of amino acids 106 to 156 ofSEQ ID NO:2.
 30. An isolated polypeptide comprising the polypeptidefragment of claim 29, and further comprising a heterologous polypeptidefused to said polypeptide fragment.
 31. The polypeptide of claim 30,wherein said heterologous polypeptide is an immunoglobulin constantregion.
 32. A composition comprising the polypeptide fragment of claim29 and a carrier.
 33. An isolated polypeptide fragment selected from thegroup consisting of: a polypeptide fragment consisting of amino acids127 to 133 of SEQ ID NO:2, and a polypeptide fragment consisting ofamino acids 142 to 150 of SEQ ID NO:2; wherein said polypeptide fragmentbinds an antibody with specificity for a polypeptide consisting of aminoacids 106 to 156 of SEQ ID NO:2.
 34. An isolated polypeptide consistingof the isolated polypeptide fragment of claim 33, and a heterologouspolypeptide fused to said polypeptide fragment.
 35. The polypeptide ofclaim 34, wherein said heterologous polypeptide comprises animmunoglobulin constant region.
 36. A composition comprising thepolypeptide fragment of claim 33 and a carrier.
 37. A method of usingthe polypeptide fragment of claim 33 produce an antibody, comprisingadministering said polypeptide fragment to an animal, and recoveringsaid antibody from said animal.
 38. The composition of claim 26, furthercomprising polyethylene glycol fused to said polypeptide fragment. 39.The composition of claim 36, further comprising polyethylene glycolfused to said polypeptide fragment.